This grant supports studies in our laboratory of the structure, function and engineering of nuclease catalysts, including T4 polynucleotide kinase, nudix hydrolases, and homing endonucleases. This latter family is of great interest, both because of their biological function as highly specific, gene-specific DNA-modification enzymes and because of their ability to 'moonlight' as highly specific RNA splicing cofactors. In this renewal, we focus on our ongoing work with homing endonucleases, describing the following aims: 1. We will determine the structure of the bifunctional l-Anil homing endonuclease/maturase bound to its cognate group I intron, and will complement these studies with biochemical analyses of its splicing activity. 2. We will determine the DNA-bound cocrystal structures of three unusual homing endonucleases that display unique departures from the 'usual' sequence-structure-function relationships for these enzymes. 3. We will engineer a group I homing endonuclease to recognize a novel target site in the XSCID linked gene encoding the IL2Ry, for the purpose of being tested as a reagent for targeted gene therapy. This aim will involve a series of basic studies and the actual engineering of the enzyme, including: a. Determination of the absolute site specificity and base pair covariance in the DNA target sites for the IAnil homing endonuclease and for H-Drel (natural and artificial homing endonucleases, respectively). b. Determination of the ability of the l-Anil and H-Drel homing endonucleases to recognize and bind potential DNA target sites in vivo (using the DamID method developed at the Hutchinson Center). c. Determination whether the affinity and cleavage activity of engineered homing endonucleases can be attenuated in a predictable manner by altering the packing and stability of the enzyme's domain interface. d. Optimization of methods for the generation and screening of combinatorial enzyme libraries. e. Generation and validation of computational methods for repacking and redesign of protein-DNA contact surfaces (collaboration with Dr. David Baker lab at University of Washington). f. Creation and testing of a novel engineered endonuclease directed towards a target in the IL2Ry gene.